The present invention relates to a novel G protein coupled receptor protein derived from the rat cerebellum and from the human brain, or salts thereof and DNA encoding the same, as well as peptides having a ligand activity to the G protein coupled receptor protein or amides or esters or salts thereof.
A variety of hormones and neurotransmitters regulate the functions in vivo through specific receptor proteins located in a cell membrane. Many of these receptor proteins mediate the transmission of intracellular signals via activation of guanine nucleotide-binding proteins (hereinafter sometimes referred to as G proteins) with which the receptor is coupled. These receptor proteins possess the common structure, i.e. seven transmembranes domains and are thus collectively referred to as G protein coupled receptors or seven-transmembrane receptors (7TMR).
G protein coupled receptor proteins exist on each functional cell surface of cells and internal organs of a living body and play very important roles as the targets of molecules, for example, hormones, neurotransmitters, physiologically active substances and the like, which molecules control, regulate or adjust the functions of cells and internal organs in the living body. These receptor proteins mediate signal transduction in a cell by binding to physiologically active substances and various reactions such as activation or inhibition of cells is caused by the thus transmitted signal.
To clarify the relation between substances which regulate complex functions in cells and internal organs of various living bodies and their specific receptor proteins, in particular, G protein coupled receptor proteins, would elucidate the functional mechanisms of cells and internal organs in various living body and thus provide a very important means for development of drugs having close relation to such functional mechanisms.
For example, in various organs of a living body, the physiological functions are controlled through regulation by many hormones, hormone-like substances, neurotransmitters or physiologically active substances. In particular, physiologically active substances present in various sites of a living body regulate its physiological functions through each of the corresponding receptor proteins. Still, there are many unknown hormones, neurotransmitters or other physiologically active substances exist in vivo and only a few receptor proteins have been reported on their structures so far. In addition, it yet remains unclear if there will be subtypes of known receptor proteins.
It is also very important for development of pharmaceuticals to clarify the relation between substances that regulate complex functions in vivo and their specific receptor proteins. Furthermore, for efficient screening of agonists and antagonists to receptor proteins in development of pharmaceuticals, it was necessary to elucidate the functions of receptor protein genes expressed in vivo and to express the genes in an appropriate expression system.
In recent years, random analysis of cDNA sequences has been actively performed as a method for analyzing genes expressed in vivo. The sequences of cDNA fragments thus obtained have been registered and published to databases as Expressed Sequence Tag (EST). However, since most EST contains only the sequence information, the function is predictable only with difficulty.
Substances that inhibit the binding of G protein coupled proteins to physiological active substances (i.e., ligands) and substances that bind to physiologically active substances thereby to induce signal transductions similar to those induced by the physiologically active substances (i.e., ligands) have been used for pharmaceuticals as antagonists or agonists specific to the receptors for regulating the biological functions. Accordingly, it is very important to discover a new G protein coupled receptor protein that is not only important for physiological expression in vivo but can be a target for developing pharmaceuticals and to clone the genes (e.g., cDNA), in search for a specific ligand, agonist, and antagonist of the novel G protein coupled receptor.
However, not all G protein coupled receptors have been found. Even now, there are many unknown G protein coupled receptors and those for which the corresponding ligands are unidentified, that is, orphan receptors. It has thus been seriously awaited to explore a novel G protein coupled receptor and clarify its function.
G Protein coupled receptors are useful in searching for a novel physiologically active substance (i.e., ligand) using the signal transduction activity as an index and in searching for agonists and antagonists of the receptor. Even if no physiological ligand is found, agonists and antagonist of the receptor may be prepared by analyzing the physiological activity of the receptor through receptor inactivation experiments (knockout animal). Ligands, agonists, and antagonists of the receptor are expected to be used as prophylactic/therapeutic and diagnostic drugs for diseases associated with dysfunction of the G protein coupled receptor.
Hypofunction or hyperfunction of the G protein coupled receptor due to genetic variation of the receptor in vivo causes some disorders in many cases. In this case, the G protein coupled receptor may be used not only for administration of antagonists or agonists of the receptor, but also for gene therapy by transfer of the receptor gene into the body (or certain specific organs) or by transfer of the antisense nucleic acid to the receptor gene. In such a gene therapy, information on the base sequence of the receptor gene is essentially required for searching of deletion or mutation in the gene. The receptor gene is also applicable as prophylactic/therapeutic and diagnostic drugs for diseases associated with dysfunction of the receptor.
In addition, finding of an endogenous ligand to the receptor or a substance having the ligand activity enables to construct the system for screening antagonists or agonists to the receptor.
The present invention provides a novel and useful G protein coupled receptor protein as described above. That is, the present invention provides a novel G protein coupled receptor protein, its partial peptides and salts thereof, as well as polynucleotides (DNA and RNA, and derivatives thereof) containing polynucleotides (DNA and RNA, and derivatives thereof) encoding the G protein coupled receptor protein and its partial peptides, recombinant vectors containing the polynucleotides, transformants bearing the recombinant vectors, methods for manufacturing the G protein coupled receptor protein and salts thereof, antibodies to the G protein coupled receptor protein, its partial peptides and salts thereof, compounds that alter the expression level of said G protein coupled receptor protein, methods for determination of ligands to the G protein coupled receptor protein, methods for screening compounds (antagonists and agonists) and salts thereof that alter the binding property of ligands and the G protein coupled receptor protein, kits for use in the screening method, compounds (antagonists and agonists) or salts thereof that alter the binding property of ligands obtainable by the screening method or obtainable using the screening kit and the G protein coupled receptor protein, and pharmaceutical compositions comprising the compounds (antagonists and agonists) that alter the binding property of ligands to the G protein coupled receptor protein, or compounds or salts thereof that alter the expression level of the G protein coupled receptor protein.
Furthermore, the present invention provides peptides having a ligand activity to the G protein coupled receptor protein.
The inventors performed extensive studies and as a result, succeeded in isolation of cDNA encoding the novel G protein coupled receptor protein derived from the rat cerebellum and from the human brain, based on the EST information prepared by the degenerated PCR method, resulting in successful analysis of the entire base sequence of the cDNA. The amino acid sequence deduced from the base sequence has supported that the first to the seventh transmembrane domains were observed on the hydrophobic plotting analysis, confirming that the protein encoded by the cDNA is a transmembrane G protein coupled receptor protein passing through the membrane seven times.
The present inventors further proceeded the investigations to explore a peptide having an intracellular Ca ion level-increasing activity to cells that express the G protein coupled receptor protein, using novel peptides found in some known peptides or on the gene data base. As a result, it has made clear that the C-terminal peptide of a protein encoded by cancer metastasis-suppressor gene KiSS-1 (Genomics, 54, 145-148, 1998) possesses an activity of activating the receptor. KiSS-1 is a protein-encoding gene. The inventors directed their attention to the sequence of the peptide consisting of 54 amino acid residues in KiSS-1 and synthesized the C-terminal partial peptides. The peptide was provided for reactivity tests with the receptor to confirm that the peptide has the ligand activity.
Peptides obtained by excision of the KiSS-1 gene products are expected to suppress tumor metastasis, since their genes are tumor metastasis-suppressor genes. Besides, the peptides are expected to play an important role in the placenta, taking into account that the gene is abundantly expressed in the placenta and hOT7T175, which is a human type receptor of the G protein coupled receptor protein, is expressed also abundantly in the placenta. Expression of the receptor is relatively abundant in human pancreas so that the peptide is expected to exert any physiological function also in the pancreas.
Based on these findings, the present inventors made extensive studies and as a result, the present invention has been accomplished.
Thus, the present invention relates to:
(1) A protein and salts thereof containing the same or substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO: 1;
(2) A protein or salts thereof according to (1), wherein the same or substantially the same amino acid sequence is represented by SEQ ID NO: 5;
(3) A partial peptide of the protein according to (1) or esters thereof or amides thereof or salts thereof;
(4) A polynucleotide containing a polynucleotide having the base sequence encoding the protein according to (1);
(5) A polynucleotide according to (4), which is DNA;
(6) A polynucleotide according to (4), which contains the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 6;
(7) A recombinant vector containing the polynucleotide according to (4);
(8) A transform ant transformed by the recombinant vector according to (7);
(9) A method for manufacturing the protein or salts thereof according to (1), which comprises culturing transformant according to (8) and producing and accumulating the protein according to (1);
(10) Antibodies to the protein or salts thereof according to (1) and to the partial peptide or esters thereof or amides thereof or salts thereof according to (3);
(11) Antibodies according to (10) which are neutralizing antibodies to inactivate signal transduction of the protein according to (1);
(12) A diagnostic composition comprising the antibodies according to (10);
(13) A ligand to the protein or salts thereof according to (1), which is obtainable using the protein or salts thereof according to (1) or using the partial peptide, esters thereof, amides thereof, or salts thereof according to (3);
(14) A pharmaceutical composition comprising the ligand according to (13);
(15) A method for determining the ligand to the protein or salts thereof according to (1), wherein the protein or salts thereof according to (1) or the partial peptide, amides thereof, esters thereof or salts thereof according to (3) are used;
(16) A method for screening a compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), which comprises using the protein or salts thereof according to (1), or the partial peptide or amides thereof, esters thereof or salts thereof according to (3);
(17) A kit for screening a compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), comprising the protein or salts thereof according to (1), the partial peptide or esters thereof, amides thereof or salts thereof according to (3) are used;
(18) A compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), which is obtainable using the screening method according to (16) or the screening kit according to (17);
(19) A pharmaceutical composition comprising the compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), which is obtainable using the screening method according to (16) or the screening kit according to (17);
(20) A method for quantifying the protein according to (1), which comprises using the antibodies according to (10);
(21) A peptide, amides thereof, esters thereof or salts thereof according to (21), which, in the amino acid sequence represented by SEQ ID NO: 10, contains a sequence of 47 to 54 amino acids from the N-terminus and comprises 8 to 54 amino acid residues;
(22) A peptide, amides thereof, ester thereof or salts thereof, which, in the amino acid sequence represented by SEQ ID NO: 10, contains the N-terminal 47-54 amino acid sequence at the C-terminus and comprises 8 to 15 amino acid residues;
(23) Amides or salts of the peptide according to (21), comprising the amino acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14;
(24) A method for screening according to (16), wherein the ligand is a peptide, amides thereof, esters thereof or salts thereof according to (21); and,
(25) A kit for screening according to (16), wherein the ligand is a peptide, amides thereof, esters thereof or salts thereof according to (21).
The present invention further provides:
(26) a protein or salts thereof according to (1) comprising: (i) an amino acid sequence represented by SEQ ID NO: 1 in which one, two, or more amino acids (preferably 1 to 30 amino acids, more preferably 1 to 9 amino acids, most preferably several (1 or 2) amino acids) are deleted; (ii) an amino acid sequence represented by SEQ ID NO: 1, to which one, two, or more amino acids (preferably 1 to 30 amino acids, more preferably 1 to 10 amino acids, most preferably several (1 or 2) amino acids) are added; (iii) an amino acid sequence represented by SEQ ID NO: 1, in which one, two, or more amino acids (preferably 1 to 30 amino acids, more preferably 1 to 10 amino acids, and most preferably several (1 or 2) amino acids) are substituted by other amino acids; and (iv) a combination of the above amino acid sequences;
(27) A method for screening according to (16), wherein comparison is made between (i) the case where the protein or salts thereof according to (1) or the partial peptide, amides thereof, esters thereof or salts thereof according to (3) are brought in contact with the ligand and (ii) the case where the protein or salts thereof according to (1) or the partial peptide, amides thereof, esters thereof or salts thereof according to (3) are brought in contact with the ligand and a test compound;
(28) A method for screening a compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), which comprises measuring and comparing (i) an amount of labeled ligand bound to the protein or salts thereof according to (1) or the partial peptide, amides thereof, esters thereof or salts thereof according to (3) where the labeled ligand is brought in contact with the protein or salts thereof according to (1) or the partial peptide, amides thereof, esters thereof or salts thereof according to (3) and (ii) an amount of labeled ligand bound to the protein or salts thereof according to (1) or the partial peptide or amides thereof, esters thereof or salts thereof according to (3) where the labeled ligand and a test compound are brought in contact with the protein or salts thereof according to (1) or the partial peptide, amides thereof, esters thereof or salts thereof according to (3);
(29) A method for screening a compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), which comprises measuring and comparing (i) an amount of labeled ligand bound to cells containing the protein according to (1) upon bringing the labeled ligand in contact with the cells, and (ii) an amount of labeled ligand bound to the cells upon bringing the labeled ligand and a test compound in contact with the cells;
(30) A method for screening a compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), which comprises measuring and comparing (i) an amount of labeled ligand bound to a membrane fraction of cells containing the protein according to (1) upon bringing the labeled ligand in contact with the cell membrane fraction, and (ii) an amount of labeled ligand bound to a membrane fraction of the cells upon bringing the labeled ligand and a test compound in contact with the cell membrane fraction;
(31) A method for screening a compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), which comprises measuring and comparing (i) an amount of labeled ligand bound to a protein expressed in cell membrane of said transformant of (8) by culturing the transformant where the labeled ligand is brought in contact with the protein expressed, and (ii) an amount of labeled ligand bound to a protein expressed in cell membrane of said transformant (8) by culturing the transformant where the labeled ligand and a test compound are brought in contact with the protein expressed;
(32) A method for screening a compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), which comprises measuring and comparing (i) a protein-mediated cell stimulating activity wherein a compound that activates the,protein or salts thereof according to (1) is brought in contact with cells containing the protein according to (1) and (ii) a protein-mediated cell stimulating activity wherein a compound that activates the protein or salts thereof according to (1) and a test compound are brought in contact with the cells containing the protein according to (1);
(33) A method for screening a compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), which comprises measuring and comparing (i) a protein-mediated cell stimulating activity wherein a compound that activates the protein or salts thereof according to (1) is brought in contact with a protein expressed in a cell membrane of the transformant according to (8) by culturing the transformant and (ii) a protein-mediated cell stimulating activity wherein a compound that activates the protein or salts thereof according to (1) and a test compound are brought in contact with a protein expressed in a cell membrane of the transformant according to (8) by culturing the transformant;
(34) A method for screening according to (32) or (33), wherein the compound that activates the protein according to (1) is a peptide, amides thereof, esters thereof or salts thereof according to (21);
(35) A compound or salts thereof that alter the binding property between a ligand and the G-protein coupled receptor protein or salts thereof according to (1), which is obtainable by the method for screening according to (27) through (34);
(36) A pharmaceutical composition comprising a compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), which is obtainable by the method for screening according to (27) through (34);
(37) A kit for screening according to (17), comprising a cell containing the protein according to (1);
(38) A kit for screening according to (17), comprising a cell membrane fraction of cell containing the protein according to (1);
(39) A kit for screening according to (17), comprising a protein expressed in a cell membrane of the transformant according to (8) by culturing the transformant;
(40) A compound or salts thereof that alter the binding property between a ligand and the protein or salts thereof according to (1), which is obtainable using the kit for screening according to (37) through (39);
(41) A pharmaceutical composition comprising a compound or salts thereof that alter the binding property between a ligand and the G-protein coupled receptor protein or salts thereof according to (1), which is obtainable using the kit for screening according to (37) through (39);
(42) A method for quantifying the protein or salts thereof according to (1), or the partial peptide, amides thereof, esters thereof or salts thereof according to (3), which comprises bringing the antibodies according to (10) in contact with the protein or salts thereof according to (1) or partial peptide, amides thereof, esters thereof or salts according to (3);
(43) A method for quantifying the protein or salts thereof according to (1), or the partial peptide, amides thereof, esters thereof or salts thereof according to (3), in a sample solution, which comprises competitively reacting the antibodies according to (10) with a sample solution and the labeled protein or salts thereof according to (1) or the labeled partial peptide, amides thereof, esters thereof or salts thereof according to (3) and, measuring ratio of the labeled protein or salts thereof according to (1) or the labeled partial peptide, amides thereof, esters thereof or salts thereof according to (3), which are bound to the antibodies; and,
(44) A method for quantifying the protein or salts thereof according to (1) or the partial peptide, amides thereof, esters thereof or salts thereof according to (3), in a sample solution, which comprises reacting a sample solution simultaneously or sequentially with antibodies of (10) immobilized on a carrier and labeled antibodies of (10) and then measuring the activity of a labeling agent on the immobilized carrier.